Method for the production of a glycosylated immunoglobulin

ABSTRACT

Herein is reported a method for the production of an immunoglobulin comprising the following steps: a) providing a eukaryotic cell comprising a nucleic acid encoding the immunoglobulin, b) cultivating the eukaryotic cell in a cultivation medium wherein the amount of glucose available in the cultivation medium per time unit is kept constant and limited to less than 80% of the amount that could maximally be utilized by the cells in the cultivation medium per time unit, and c) recovering the immunoglobulin from the culture.

RELATED APPLICATION

This is a continuation application which claims priority under 35 USC §120 to non-provisional application Ser. No. 12/911,300, filed Oct. 25,2010, which claims priority to European application no. 09013455.2 filedon Oct. 26, 2009, the contents of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Herein is reported a method in the field of immunoglobulin production incells, whereby the glycosylation pattern of the produced immunoglobulincan be modified based on the cultivation conditions.

In recent years the production of immunoglobulins has steadily increasedand it is likely that immunoglobulins will become the biggest group oftherapeutics available for the treatment of various diseases in the nearfuture. The impact of immunoglobulins emerges from their specificity,which comprises their specific target recognition and binding functionas well as the activation of specific effects concurrently with or afterantigen/Fc-receptor binding.

The specific target recognition and binding is mediated by the variableregion of the immunoglobulin. Other parts of the immunoglobulinmolecule, from which effects originate, are posttranslationalmodifications, such as the glycosylation pattern. The posttranslationalmodifications do have an influence on the efficacy, stability,immunogenic potential, binding etc. of an immunoglobulin. In connectiontherewith complement-dependent cytotoxicity (CDC), antibody-dependentcellular cytotoxicity (ADCC) and induction of apoptosis have to beaddressed.

It has been reported that the glycosylation pattern of immunoglobulins,i.e. the saccharide composition and number of attached glycostructures,has a strong influence on the biological properties (see e.g. Jefferis,R., Biotechnol. Prog. 21 (2005) 11-16). Immunoglobulins produced bymammalian cells contain 2-3% by mass carbohydrates (Taniguchi, T., etal., Biochem. 24 (1985) 5551-5557). This is equivalent e.g. in animmunoglobulin of class G (IgG) to 2.3 oligosaccharide residues in anIgG of mouse origin (Mizuochi, T., et al., Arch. Biochem. Biophys. 257(1987) 387-394) and to 2.8 oligosaccharide residues in an IgG of humanorigin (Parekh, R. B., et al., Nature 316 (1985) 452-457), whereofgenerally two are located in the Fc-region and the remaining in thevariable region (Saba, J. A., et al., Anal. Biochem. 305 (2002) 16-31).

In the Fc-region of an immunoglobulin of class G oligosaccharideresidues can be introduced via N-glycosylation at amino acid residue297, which is an asparagine residue (denoted as Asn²⁹⁷). Youings et al.have shown that a further N-glycosylation site exists in 15% to 20% ofpolyclonal IgG molecules in the Fab-region (Youings, A., et al.,Biochem. J., 314 (1996) 621-630; see e.g. also Endo, T., et al., Mol.Immunol. 32 (1995) 931-940). Due to inhomogeneous, i.e. asymmetric,oligosaccharide processing, multiple isoforms of an immunoglobulin withdifferent glycosylation pattern exist (Patel, T. P., et al., Biochem. J.285 (1992) 839-845; Ip, C. C., et al., Arch. Biochem. Biophys. 308(1994) 387-399; Lund, J., et al., Mol. Immunol. 30 (1993) 741-748).Concurrently the structure and distribution of the oligosaccharides isboth highly reproducible (i.e. non-random) and site specific (Dwek, R.A., et al., J. Anat. 187 (1995) 279-292).

Some characteristics of an immunoglobulin are directly linked to theglycosylation of the Fc-region (see e.g. Dwek, R. A., et al., J. Anat.187 (1995) 279-292; Lund, J., et al., J. Immunol. 157 (1996) 4963-4969;Lund, J., FASEB J. 9 (1995) 115-119; Wright, A. and Morrison, S. L., J.Immunol. 160 (1998) 3393-3402), such as for example thermal stabilityand solubility (West, C. M., Mol. Cell. Biochem. 72 (1986) 3-20),antigenicity (Turco, S. J., Arch. Biochem. Biophys. 205 (1980) 330-339),immunogenicity (Bradshaw, J. P., et al., Biochim. Biophys. Acta 847(1985) 344-351; Feizi, T. and Childs, R. A., Biochem. J. 245 (1987)1-11; Schauer, R., Adv. Exp. Med. Biol. 228 (1988) 47-72), clearancerate/circulatory half-life (Ashwell, G. and Harford, J., Ann. Rev.Biochem. 51 (1982) 531-554; McFarlane, I. G., Clin. Sci. 64 (1983)127-135; Baenziger, J. U., Am. J. Path. 121 (1985) 382-391; Chan, V. T.and Wolf, G., Biochem. J. 247 (1987) 53-62; Wright, A., et al.,Glycobiology 10 (2000) 1347-1355; Rifai, A., et al., J. Exp. Med. 191(2000) 2171-2182; Zukier, L. S., et al., Cancer Res. 58 (1998)3905-3908), and biological specific activity (Jefferis, R. and Lund, J.,in Antibody Engineering, ed. by Capra, J. D., Chem. Immunol. Basel,Karger, 65 (1997) 111-128).

Factors influencing the glycosylation pattern have been investigated,such as for example presence of fetal calf serum in the fermentationmedium (Gawlitzek, M., et al., J. Biotechnol. 42(2) (1995) 117-131),buffering conditions (Mülthing, J., et al., Biotechnol. Bioeng. 83(2003) 321-334), dissolved oxygen concentration (Saba, J. A., et al.,Anal. Biochem. 305 (2002) 16-31; Kunkel, J. P., et al., J. Biotechnol.62 (1998) 55-71; Lin, A. A., et al., Biotechnol. Bioeng. 42 (1993)339-350), position and conformation of the oligosaccharide as well ashost cell type and cellular growth state (Hahn, T. J. and Goochee, C.F., J. Biol. Chem. 267 (1992) 23982-23987; Jenkins, N., et al., Nat.Biotechnol. 14 (1996) 975-981), cellular nucleotide-sugar metabolism(Hills, A. E., et al., Biotechnol. Bioeng. 75 (2001) 239-251), nutrientlimitations (Gawlitzek, M., et al., Biotechnol. Bioeng. 46 (1995)536-544; Hayter, P. M., et al., Biotechnol. Bioeng. 39 (1992) 327-335),especially glucose restriction (Tachibana, H., et al., Cytotechnology 16(1994) 151-157), and extracellular pH (Borys, M. C., et al.,Bio/Technology 11 (1993) 720-724).

Increased oligomannose structures as well as truncated oligosaccharidestructures have been observed by the recombinant expression ofimmunoglobulins e.g. in NS0 myeloma cells (Ip, C. C., et al., Arch.Biochem. Biophys. 308 (1994) 387-399; Robinson, D. K., et al.,Biotechnol. Bioeng. 44 (1994) 727-735). Under glucose starvationconditions variations in glycosylation, such as attachment of smallerprecursor oligosaccharides or complete absence of oligosaccharidemoieties, have been observed in CHO cells, Murine 3T3 cells, rathepatoma cells, rat kidney cells and Murine myeloma cells (Rearick, J.I., et al., J. Biol. Chem. 256 (1981) 6255-6261; Davidson, S. K. andHunt, L. A., J. Gen. Virol. 66 (1985) 1457-1468; Gershman, H. andRobbins, P. W., J. Biol. Chem. 256 (1981) 7774-7780; Baumann, H. andJahreis, G. P., J. Biol. Chem. 258 (1983) 3942-3949; Strube, K.-H., etal., J. Biol. Chem. 263 (1988) 3762-3771; Stark, N. J. and Heath, E. C.,Arch. Biochem. Biophys. 192 (1979) 599-609). A strategy based on lowglutamine/glucose concentrations was reported by Wong, D. C. F., et al.,Biotechnol. Bioeng. 89 (2005) 164-177.

The Japanese Patent Application JP 62-258252 reports a perfusion cultureof mammalian cells, whereas U.S. Pat. No. 5,443,968 reports a fed-batchculture method for protein secreting cells. In WO 98/41611 a method forcultivating cells is reported effective to adapt the cells to ametabolic state characterized by low lactate production. A method forculturing cells in order to produce substances is reported in WO2004/048556. Elbein, A. D., Ann. Rev. Biochem. 56 (1987) 497-534,reports that mammalian cells when incubated in the absence of glucosetransfer mannose-5 containing structures instead of mannose-9 containingstructures to proteins. The dependence of pCO2 influences during glucoselimitation on CHO cell growth, metabolism and IgG production is reportedby Takuma, S., et al. in Biotechnol. Bioeng. 97 (2007) 1479-1488.

SUMMARY OF THE INVENTION

It has been found that the amount of the mannose-5 glycostructure in theglycosylation pattern of a polypeptide produced by a eukaryotic cell canbe modified based on the amount of glucose provided to the cell in thecultivation process. By reducing the amount of glucose available, e.g.by changing the DGL value from 1.0 to smaller values of e.g. 0.8, 0.6,0.5, 0.4, or 0.2, a modification in the mannose-5 glycostructure amountin the glycosylation pattern can be obtained. The DGL value orrespectively the amount of glucose available per time unit has to bekept constant and at a defined reduced value per time unit.

A first aspect as reported herein is a method for the production of apolypeptide, in one embodiment of an immunoglobulin, in a eukaryoticcell, comprising the following steps

-   -   a) providing a eukaryotic cell comprising a nucleic acid        encoding the polypeptide,    -   b) cultivating the cell under conditions wherein the degree of        glucose limitation (DGL) is kept constant and wherein the DGL is        less than 0.8, and    -   c) recovering the polypeptide from the culture,        wherein the fraction of the polypeptide with a mannose-5        glycostructure is 10% or less of the sum comprising the amount        of the polypeptide with a mannose-5 glycostructure, the amount        of the polypeptide G(0) isoform, the amount of the polypeptide        G(1) isoform, and the amount of the polypeptide G(2) isoform.

In one embodiment the DGL is kept constant in the range from 0.8 to 0.2.In a further embodiment the DGL is kept constant in the range from 0.6to 0.4. In another embodiment the fraction of the polypeptide with amannose-5 glycostructure is 8% or less of the sum comprising thepolypeptide with a mannose-5 glycostructure, the polypeptide G(0)isoform, the polypeptide G(1) isoform, and the polypeptide G(2) isoform.In still another embodiment the polypeptide is an immunoglobulin, in oneembodiment an immunoglobulin of class G or E.

Another aspect as reported herein is a method for the production of animmunoglobulin comprising the following steps:

-   -   a) providing a mammalian cell comprising a nucleic acid encoding        the immunoglobulin,    -   b) cultivating the cell in a cultivation medium wherein the        amount of glucose available in the cultivation medium per time        unit is kept constant and limited to less than 80% of the amount        that could maximally be utilized by the cells in the cultivation        medium per time unit, and    -   c) recovering the immunoglobulin from the cells or the        cultivation medium.

In one embodiment the amount of glucose available in the cultivationmedium per time unit is kept constant and limited to a value in therange from 80% to 20%. In a further embodiment the range is from 60% to40%. In another embodiment the cells in the cultivation medium are theviable cells in the cultivation medium.

In one embodiment of the aspects as reported herein the eukaryotic cellis selected from CHO cells, NS0 cells, HEK cells, BHK cells, hybridomacells, PER.C6® cells, insect cells, or Sp2/0 cells. In one embodimentthe eukaryotic cell is a Chinese Hamster Ovary (CHO) cell. In anotherembodiment of the aspects as reported herein the cultivating is at a pHvalue in the range from about pH 7.0 to about pH 7.2.

In still another embodiment of the aspects as reported herein thecultivating is a continuous or a fed-batch cultivating. The methods maycomprise in another embodiment a final step of purifying thepolypeptide. In still another embodiment the cell is cultivated for sixto twenty days or for six to fifteen days. In a further embodiment thecell is cultivated for six to eight days.

Another aspect as reported herein is a composition comprising animmunoglobulin, wherein the composition has been prepared with a methodas reported herein.

In one embodiment the immunoglobulin is an anti-IL-6R antibody. In afurther embodiment the anti-IL-6R antibody comprises Tocilizumab. Inanother embodiment the mannose-5 glycostructure attached to theanti-IL-6R antibody is 8% or less. In still a further embodiment themannose-5 glycostructure is 6% or less. In another embodiment themannose-5 glycostructure is 4% or less.

The invention also concerns a composition comprising an antibody thatbinds human interleukin 6 receptor (anti-IL-6R antibody) witholigosaccharide attached thereto, wherein mannose-5 glycostructure (M5)content in the composition is 8% or less, e.g. less than 5%, forexample, 4% or less. In one embodiment, the anti-IL-6R antibody isTocilizumab and/or has been produced by a recombinant Chinese HamsterOvary (CHO) cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B Viable cell density (FIG. 1A) and cell viabilityprofiles (FIG. 1B) in the fed-batch mode using the DGL control; opencircle: initial cell density of 8×10⁵ cells/ml; filled triangle: initialcell density of 10×10⁵ cells/ml; open square: initial cell density of12×10⁵ cells/ml.

FIG. 2 Time courses of DGL in the fed-batch mode in immunoglobulinproduction; circle: initial cell density of 8×10⁵ cells/ml; triangle:initial cell density of 10×10⁵ cells/ml; square: initial cell density of12×10⁵ cells/ml.

FIG. 3 Feeding profiles based on DGL by the fed-batch mode inimmunoglobulin production; circles: initial cell density of 8×10⁵cells/ml; triangle: initial cell density of 10×10⁵ cells/ml; square:initial cell density of 12×10⁵ cells/ml.

FIG. 4 Immunoglobulin production profiles by the fed-batch mode in theDGL control; open circles: initial cell density of 8×10⁵ cells/ml;filled triangle: initial cell density of 10×10⁵ cells/ml; open square:initial cell density of 12×10⁵ cells/ml; filled small circle: constantfeeding method: FR=0.02 g glucose/h (control)

FIG. 5 Time curse of DGL during a fed-batch cultivation of a cell:diamond: single feed daily feeding, square: dual feed daily feeding;triangle: single feed profile feeding; X: dual feed profile feeding.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a method for the production of an immunoglobulincomprising the following steps:

-   -   a) cultivating a mammalian cell comprising a nucleic acid        encoding the immunoglobulin in a cultivation medium at a        constant DGL of less than 0.8 (i.e. the amount of glucose        available per time unit is constant and 80% or less of the        amount of glucose that can maximally be utilized by the cell per        time unit), and    -   b) recovering the immunoglobulin from the cells or the culture        medium.

With the method as reported herein an immunoglobulin can be obtainedwherein the amount of the immunoglobulin with a mannose-5 glycostructuredepends on the adjusted DGL value, and wherein the amount is thefraction of the sum of the amount of the immunoglobulin with a mannose-5glycostructure, and of the immunoglobulin G(0) isoform, and of theimmunoglobulin G(1) isoform, and of the immunoglobulin G(2) isoform. Inone embodiment the DGL is from 0.8 to 0.2. In this embodiment thefraction is 10% or less. In another embodiment the DGL is from 0.6 to0.4. In this embodiment the fraction is 6% or less. With the method asreported herein an immunoglobulin can be obtained wherein the fractionof the immunoglobulin having a mannose-5 glycostructure is 10% or lessof the sum comprising the amount of the immunoglobulin with a mannose-5glycostructure, the amount of the immunoglobulin G(0) isoform, theamount of the immunoglobulin G(1) isoform, and the amount of theimmunoglobulin G(2) isoform. In another embodiment the fraction is thearea-% fraction determined in a liquid chromatography method. In oneembodiment the DGL is maintained in the range from 0.8 to 0.2. Inanother embodiment the DGL is maintained in the range from 0.6 to 0.2.In still another embodiment the DGL is maintained in the range from 0.6to 0.4. In one embodiment the amount of glucose that can maximally beutilized by the cell per time unit is the average amount of glucose thatis utilized in a cultivation in which all compounds are available inexcess, i.e. no compound is limiting the growth of the cell, determinedbased on at least five cultivations. In one embodiment the fraction isdetermined on day seven of the cultivation.

Methods and techniques known to a person skilled in the art, which areuseful for carrying out the current invention, are described e.g. inAusubel, F. M. (ed.), Current Protocols in Molecular Biology, Volumes Ito III (1997), Wiley and Sons; Sambrook, J., et al., Molecular Cloning:A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (2001); Glover, N. D. (ed.), DNA Cloning: APractical Approach, Volumes I and II (1985); Freshney, R. I. (ed.),Animal Cell Culture (1986); Miller, J. H. and Calos, M. P. (eds.), GeneTransfer Vectors for Mammalian Cells, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1987); Watson, J. D., et al.,Recombinant DNA, Second Edition, N.Y., W.H. Freeman and Co (1992);Winnacker, E. L., From Genes to Clones, N.Y., VCH Publishers (1987);Celis, J. (ed.), Cell Biology, Second Edition, Academic Press (1998);Freshney, R. I., Culture of Animal Cells: A Manual of Basic Techniques,Second Edition, Alan R. Liss, Inc., N.Y. (1987).

The use of recombinant DNA technology enables the production of numerousderivatives of a polypeptide. Such derivatives can, for example, bemodified in individual or several amino acid positions by substitution,alteration or exchange. The derivatization can, for example, be carriedout by means of site directed mutagenesis. Such variations can easily becarried out by a person skilled in the art (Sambrook, J., et al.,Molecular Cloning: A laboratory manual, Third Edition (2001) Cold SpringHarbor Laboratory Press, New York, USA; Hames, B. D. and Higgins, S. G.,Nucleic acid hybridization—a practical approach (1985) IRL Press,Oxford, England).

The term “nucleic acid” denotes a naturally occurring or partially orfully non-naturally occurring nucleic acid molecule encoding apolypeptide. The nucleic acid can be build up of DNA-fragments which areeither isolated or synthesized by chemical means. The nucleic acid canbe integrated into another nucleic acid, e.g. in an expression plasmidor the genome/chromosome of a eukaryotic cell. The term “plasmid”includes shuttle and expression plasmids. Typically, the plasmid willalso comprise a prokaryotic propagation unit comprising an origin ofreplication (e.g. the ColEl origin of replication) and a selectablemarker (e.g. ampicillin or tetracycline resistance gene), forreplication and selection, respectively, of the plasmid in prokaryoticcells. To a person skilled in the art procedures and methods are wellknown to convert an amino acid sequence, e.g. of a polypeptide, into acorresponding nucleic acid encoding the respective amino acid sequence.Therefore, a nucleic acid is characterized by its nucleic acid sequenceconsisting of individual nucleotides and likewise by the amino acidsequence of a polypeptide encoded thereby.

The term “expression cassette” denotes a nucleic acid that contains theelements necessary for expression and optionally for secretion of atleast the contained structural gene in/from a cell, such as a promoter,polyadenylation site, and 3′- and 5′-untranslated regions.

The term “gene” denotes e.g. a segment on a chromosome or on a plasmid,which is necessary for the expression of a polypeptide. Beside thecoding region a gene comprises other functional elements including apromoter, introns, and one or more transcription terminators. A“structural gene” denotes the coding region of a gene without a signalsequence.

The term “expression” denotes the transcription and translation of astructural gene within a cell. The level of transcription of astructural gene in a cell can be determined on the basis of the amountof corresponding mRNA that is present in the cell. For example, mRNAtranscribed from a selected nucleic acid can be quantitated by PCR or byNorthern hybridization (see e.g. Sambrook et al. (supra)). A polypeptideencoded by a nucleic acid can be quantitated by various methods, e.g. byELISA, by determining the biological activity of the polypeptide, or byemploying methods that are independent of such activity, such as Westernblotting or radioimmunoassay, using antibodies that recognize and bindto the polypeptide (see e.g. Sambrook et al. (supra)).

The term “cell” denotes a cell into which a nucleic acid encoding apolypeptide, in one embodiment a heterologous polypeptide, has beenintroduced. The term “cell” includes both prokaryotic cells used forpropagation of plasmids/vectors as well as eukaryotic cells used forexpression of the structural gene. In one embodiment a eukaryotic cellfor the expression of an immunoglobulin is a mammalian cell. In anotherembodiment the mammalian cell is selected from CHO cells, NS0 cells,Sp2/0 cells, COS cells, HEK cells, BHK cells, PER.C6® cells, andhybridoma cells. A eukaryotic cell can be selected in addition frominsect cells, such as caterpillar cells (Spodoptera frugiperda, sfcells), fruit fly cells (Drosophila melanogaster), mosquito cells (Aedesaegypti, Aedes albopictus), and silkworm cells (Bombyx Mori), and thelike.

The term “polypeptide” denotes a polymer of amino acid residues joinedby peptide bonds, whether produced naturally or synthetically.Polypeptides of less than about 20 amino acid residues may be referredto as “peptides”. Polypeptides of more than 100 amino acid residues orcovalent and non-covalent aggregates comprising more than onepolypeptide may be referred to as “proteins”. Polypeptides may comprisenon-amino acid components, such as carbohydrate groups. The non-aminoacid components may be added to the polypeptide by the cell in which thepolypeptide is produced, and may vary with the type of cell.Polypeptides are defined herein in terms of their amino acid sequence inN- to C-terminal direction. Additions thereto, such as carbohydrategroups, are generally not specified, but may be present nonetheless.

The term “heterologous DNA” or “heterologous polypeptide” denotes a DNAmolecule or a polypeptide, or a population of DNA molecules or apopulation of polypeptides, which do not exist naturally within a givencell. DNA molecules heterologous to a particular cell may contain DNAderived from the cell's species (i.e. endogenous DNA) so long as thatDNA is combined with non-host DNA (i.e. exogenous DNA). For example, aDNA molecule containing a non-cell's DNA segment, e.g. encoding apolypeptide, operably linked to a cell's DNA segment, e.g. comprising apromoter, is considered to be a heterologous DNA molecule. Likewise, aheterologous DNA molecule can comprise an endogenous structural geneoperably linked to an exogenous promoter. A polypeptide encoded by aheterologous DNA molecule is a “heterologous” polypeptide.

The term “expression plasmid” denotes a nucleic acid comprising at leastone structural gene encoding a polypeptide to be expressed. Typically,an expression plasmid comprises a prokaryotic plasmid propagation unit,including an origin of replication and a selection marker, e.g. for E.coli, an eukaryotic selection marker, and one or more expressioncassettes for the expression of the structural gene(s) of interest eachin turn comprising a promoter, at least one structural gene, and atranscription terminator including a polyadenylation signal. Geneexpression is usually placed under the control of a promoter, and such astructural gene is to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

The term “isolated polypeptide” denotes a polypeptide that isessentially free from associated cellular components, such ascarbohydrate, lipid, or other proteinaceous or non-proteinaceousimpurities, which are not covalently associated with the polypeptide.Typically, a preparation of an isolated polypeptide contains in certainembodiments the polypeptide in a highly purified form, i.e. at leastabout 80% pure, at least about 90% pure, at least about 95% pure,greater than 95% pure, or greater than 99% pure. One way to show that aparticular protein preparation contains an isolated polypeptide is bythe appearance of a single band following sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-page) of the preparation andCoomassie Brilliant Blue staining of the gel. However, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers, or alternativelyglycosylated or derivatized forms.

Immunoglobulins in general are assigned into five different classes: IgA(immunoglobulin of class A), IgD, IgE, IgG and IgM. Between theseclasses the immunoglobulins differ in their overall structure and/oramino acid sequence but have the same building blocks. Completeimmunoglobulins are built up of two pairs of polypeptide chains, eachcomprising an immunoglobulin light polypeptide chain (short: lightchain) and an immunoglobulin heavy polypeptide chain (short: heavychain). In turn the chains comprise a variable region and a constantregion. In a light chain both regions consist of one domain, whereas ina heavy chain the variable region consists of one domain and theconstant region comprises up to five domains (in N- to C-terminaldirection): the C_(H)1-domain, optionally the hinge region domain, theC_(H)2-domain, the C_(H)3-domain, and optionally the C_(H)4-domain. Animmunoglobulin can be dissected in a Fab- and an Fc-region. The entirelight chain, the heavy chain variable domain and the C_(H)1 domain arereferred to as Fab-region (fragment antigen binding-region). TheFc-region comprises the C_(H)2-, C_(H)3-, and optionally theC_(H)4-domain.

As used herein, the term “immunoglobulin” denotes a protein consistingof one or more polypeptides. The encoding immunoglobulin genes includethe different constant region genes as well as the myriad immunoglobulinvariable region genes. The term “immunoglobulin” comprise in oneembodiment monoclonal antibodies and fragments thereof, such as anisolated heavy chain, or a heavy chain constant region, as well asfusion polypeptides comprising at least an immunoglobulin heavy chainC_(H)2-domain. In one embodiment of the method as reported herein theimmunoglobulin is a complete immunoglobulin, in another embodiment theimmunoglobulin is an Fc-region of a complete immunoglobulin. In anotherembodiment the immunoglobulin is an immunoglobulin, or an immunoglobulinfragment, or an immunoglobulin conjugate.

The term “immunoglobulin fragment” denotes a polypeptide comprising atleast the C_(H)2-domain of an immunoglobulin delta, epsilon, or alphaheavy chain, and/or the C_(H)3-domain of an immunoglobulin epsilon ordelta heavy chain. Encompassed are also derivatives and variants thereofwherein the N-glycosylation motif Asn-Xaa-Ser/Thr in the C_(H)2- orC_(H)3-domain is not changed.

The term “immunoglobulin conjugate” denotes a polypeptide comprising atleast the C_(H)2-domain of an immunoglobulin delta, epsilon, or alphaheavy chain, and/or the C_(H)3-domain of an immunoglobulin epsilon ordelta heavy chain fused to a non-immunoglobulin polypeptide. Therein theN-glycosylation motif Asn-Xaa-Ser/Thr in the C_(H)2- or C_(H)3-domain isnot changed.

The oligosaccharides attached to Asn²⁹⁷ (IgG, IgE) or Asn²⁶³ (IgA) of aC_(H)2-domain and/or to Asn³⁹⁴, Asn⁴⁴⁵, or Asn⁴⁹⁶ (IgE, IgD) of aC_(H)3-domain of an immunoglobulin heavy chain have a biantennarystructure (Mizuochi, T., et al., Arch. Biochem. Biophys. 257 (1987)387-394), i.e. they consist of a core structure ofMan(α1-4)GlcNAc(β1-4)GlcNAc→Asnwith an optional Fuc(α1-6) linkage at the terminal GlcNAc residue. Twoouter-arms are connected to the terminal mannose of the core structurehaving the formulaGal(β1-4)GlcNAc(β1-2)Man(α1-6)→Man, andGal(β1-4)GlcNAc(β1-2)Man(α1-3)→Man,wherein the terminal galactose residues are optional (Man=mannose,GlcNAc=N-acetyl glucose, Gal=galactose; Fuc=fucose).

TABLE 1 Glycosylation sites of immunoglobulins. immunoglobulin classresidue to which a glycostructure can be attached IgG Asn 297 IgE Asn255, Asn 297, Asn 361, Asn 371, Asn 394 IgA Asn 263, Asn 459 IgD Asn445, Asn 496 IgM Asn 395

The term “the amount of the immunoglobulin G(0) isoform, the amount ofthe immunoglobulin G(1) isoform, and the amount of the immunoglobulinG(2) isoform” denotes the sum of the amounts of the different,heterogeneous, biantennary oligosaccharides N-linked to an asparagine(Asn) of an immunoglobulin. The G(2) isoform has a terminal galactoseresidue on each of the outer-arms of the oligosaccharide structure, theG(1) isoform bears only a galactose residue on either the (α1-6) or(α1-3) linked outer-arm, and the G(0) isoform bears no galactose residueon both outer-arms.

The term “mannose-5 glycostructure” denotes an oligomannose-structurelinked to an Asn residue of a polypeptide comprising or consisting offive mannose residues and two N-acetyl glucose core residues, forming atriantennary structure.

One aspect as reported herein is a method for the production of animmunoglobulin comprising the following steps:

-   -   a) cultivating a eukaryotic cell, preferably a mammalian cell,        comprising one or more nucleic acid(s) encoding the        immunoglobulin in a cultivation medium wherein the amount of        glucose available in the cultivation medium per time unit is        kept constant and limited to a value of less than 80% of the        amount that could maximally be utilized by the eukaryotic cells        in the cultivating per time unit, and    -   b) recovering the immunoglobulin from the cell or the culture        medium and thereby producing an immunoglobulin.

With this method an immunoglobulin is obtained comprising at most 10% ofan immunoglobulin with a mannose-5 glycostructure. The 10% arecalculated based on the sum of the amount of the immunoglobulin with amannose-5 glycostructure, the amount of the immunoglobulin G(0) isoform,the amount of the immunoglobulin G(1) isoform, and the amount of theimmunoglobulin G(2) isoform.

The terms “degree of glucose limitation” and its abbreviation “DGL”,which can be used interchangeably herein, denote the ratio of thecurrent specific glucose consumption rate of a single cell in acultivation to the maximum known specific glucose consumption rate ofthe single cell or a single cell of the same kind. The degree of glucoselimitation is defined as

${D\; G\; L} = \frac{qGlc}{{qGlc}_{\max}}$

-   with qGlc=current specific glucose consumption rate of a single    cell;    -   qGlc_(max)=maximum known specific glucose consumption rate for        this single cell or a single cell of the same kind.

The DGL can vary between DGL_(maintenance) and 1 wherebyDGL_(maintenance) (<1 and >0) denotes complete growth limitation and 1denotes no limitation or complete glucose excess.

The introduction of glycostructures to polypeptides, e.g.immunoglobulins, is a post-translational modification. Due toincompleteness of the glycosylation procedure of the respective cellevery expressed polypeptide is obtained with a glycosylation patterncomprising different glycostructures. Thus, a polypeptide is obtainedfrom a cell expressing it in form of a composition comprisingdifferently glycosylated forms of the same polypeptide, i.e. with thesame amino acid sequence. The sum of the individual glycostructures isdenoted as glycosylation pattern, comprising e.g. polypeptides withcompletely missing glycostructures, differently processedglycostructures, and/or differently composed glycostructures.

One glycostructure is the mannose-5 glycostructure (also denoted ashigh-mannose, Man5, M5, or oligo-mannose). It has been reported, thatthe fraction of recombinantly produced polypeptides with the mannose-5glycostructure is increased with prolonged cultivation time or underglucose starvation conditions (Robinson, D. K., et al., Biotechnol.Bioeng. 44 (1994) 727-735; Elbein, A. D., Ann. Rev. Biochem. 56 (1987)497-534).

It has been found that the amount of the mannose-5 glycostructure in theglycosylation pattern of a polypeptide produced by a eukaryotic cell canbe modified based on the amount of glucose provided to the cell in thecultivation process. It has been found that by reducing the amount ofglucose, i.e. by changing the DGL value from 1.0 to smaller values ofe.g. 0.8, 0.6, 0.5, 0.4, or 0.2, a modification in the mannose-5glycostructure amount in the glycosylation pattern can be achieved. Inone embodiment the DGL value is kept constant at a value within a range,such as from 0.8 to 0.2, or from 0.6 to 0.4. That is, the production ofa polypeptide, in one embodiment of an immunoglobulin, can be performedunder conditions wherein a restricted amount of glucose is available tothe cultivated cell in order to obtain the polypeptide with a definedamount of the mannose-5 glycostructure in the glycosylation pattern. Ithas been found that a cultivation with an amount of glucose availableper time unit of 80% or less of the amount of glucose that can maximallybe utilized by the cells per time unit, in one embodiment byexponentially growing cells, i.e with a DGL of 0.8 or less, yields apolypeptide with a glycosylation pattern in which the amount of themannose-5 glycostructure is changed compared to a cultivation with a DGLof 1.0. In one embodiment the cell density is the viable cell density.Additionally the obtained polypeptide yield is increased.

The term “the amount of glucose that can maximally be utilized by thecell per time unit” denotes the amount of glucose that is maximallyconsumed or utilized or metabolized per time unit by a single cell underoptimum growth conditions in the exponential growth phase in acultivation without any nutrient limitation. Thus, the amount of glucosethat can maximally be utilized by the cell per time unit can bedetermined by determining the amount of glucose that is metabolized pertime unit by a cell under optimum growth conditions in the exponentialgrowth phase in a cultivation without any nutrient limitation. A furtherincrease of the available amount of glucose will not further increase,i.e. change, the amount of glucose that can maximally be utilized by thecell per time unit. This amount defines the maximum level of glucoseconsumption of a single cell. This does not denote that a geneticallymodified version of the cell might not have an even higher maximum levelof glucose consumption. Alternatively the amount of glucose that can bemaximally be utilized by the cell per time unit can be determined basedon previous cultivations and the monitored data.

The process as reported herein is particularly simple to carry out,associated with a minimum effort for measuring and control, andparticularly economic.

Without restrictions, e.g. insufficient nutrient supply, cultivatedcells grow and consume nutrients at maximum rates in an uneconomicmanner. One of the consumed culture medium nutrients is glucose, whichis metabolized by the cultivated cells in order to produce energy andbuilding blocks for the cell's metabolism. In the presence of excessglucose the cell's metabolism is running at the maximum turnover ratefor glucose. The amount of glucose that can maximally be utilized by thecell per time unit can for example be determined from the glucoseconsumption of exponentially growing cells in the presence of excessglucose cultivated with or under the same cultivation conditions thatwill also be used in the cultivation with restricted glucose, i.e. withan amount of glucose available per time unit that is smaller than thatwhich can be utilized by the cell. This maximum amount can be calculatedeasily by determining the cell density and glucose concentration at thebeginning and end of a fixed time range. The value is normally in arange from 0.006 to 190 mmol/hour/10⁹ cells (Baker, K. N., et al.,Biotechnol. Bioeng. 73 (2001) 188-202; WO 98/41611; Müthing, J., et al.,Biotechnol. Bioeng. 83 (2003) 321-334; WO 2004/048556). In oneembodiment the qGlc_(max) is about 0.142 mmol/hour/10⁹ cells understandard process conditions at pH 7.0.

The method as reported herein is performed in one embodiment underconditions wherein the amount of glucose available per time unit is keptconstant and at 80% or less of the amount of glucose that can maximallybe utilized by the cell per time unit (0.8≥DGL>0), in one embodiment theamount of glucose available is kept constant and at 60% or less(0.6≥DGL>0), in another embodiment at 50% or less (0.5≥DGL>0), and instill another embodiment at about 40%. The term “about” as used withinthis application denotes that the value is no exact value it is merelythe central point of a range wherein the value can vary up to 10%, i.e.the term “about 40%” denotes a range from 44% to 36% (DGL=0.44−0.36).

In one embodiment the cultivating is with an amount of glucose availableper time unit that is kept constant in a range between 80% and 10% ofthe amount of glucose that can maximally be utilized by the cell pertime unit (0.8≥DGL≥0.1). In another embodiment the amount of glucoseavailable is kept constant in a range between 60% and 10% (0.6≥DGL≥0.1).In a further embodiment the amount of glucose available is kept constantin a range between 50% and 10% (0.5≥DGL≥0.1). In another embodiment theamount of glucose available is kept constant in a range between 45% and20% (0.45≥DGL≥0.2). In also an embodiment the amount of glucoseavailable is kept between 80% and 60% (0.8≥DGL≥0.6).

In one embodiment the method comprises the step of cultivating the cellunder conditions wherein the DGL is kept constant and at a value ofabout 0.4, whereby the cultivating comprises starting with a DGL between1.0 and 0.5, lowering the DGL to a value of about 0.4, and keeping theDGL constant thereafter. In one embodiment the lowering of the DGL iswithin a time period of 100 hours. The term “keeping the DGL constant”and grammatical equivalents thereof denote that the DGL value ismaintained during a time period, i.e. the variation of the DGL value iswithin 10% of the value (see e.g. FIG. 2).

The immunoglobulin is recovered after production, either directly orafter disintegration of the cell. The recovered immunoglobulin is in oneembodiment purified with a method known to a person skilled in the art.Different methods are well established and widespread used for proteinpurification, such as affinity chromatography with microbial proteins(e.g. protein A or protein G affinity chromatography), ion exchangechromatography (e.g. cation exchange (carboxymethyl resins), anionexchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

For example, a purification process for immunoglobulins in generalcomprises a multistep chromatographic part. In the first stepnon-immunoglobulin polypeptides are separated from the immunoglobulinfraction by an affinity chromatography, e.g. with protein A or G.Afterwards, e.g., ion exchange chromatography can be performed todisunite the individual immunoglobulin classes and to remove traces ofprotein A, which has been coeluted from the first column. Finally achromatographic step is employed to separate immunoglobulin monomersfrom multimers and fragments of the same class.

General chromatographic methods and their use are known to a personskilled in the art. See for example, Chromatography, 5^(th) edition,Part A: Fundamentals and Techniques, Heftmann, E. (ed.), ElsevierScience Publishing Company, New York, (1992); Advanced Chromatographicand Electromigration Methods in Biosciences, Deyl, Z. (ed.), ElsevierScience BV, Amsterdam, The Netherlands, (1998); Chromatography Today,Poole, C. F. and Poole, S. K., Elsevier Science Publishing Company, NewYork, (1991); Scopes, R. K., Protein Purification: Principles andPractice (1982); Sambrook, J., et al. (ed.), Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 2001; or Current Protocols in MolecularBiology, Ausubel, F. M., et al. (eds), John Wiley & Sons, Inc., New York(1990).

In one embodiment the recovered immunoglobulin is characterized by theamount of the immunoglobulin having a mannose-5 glycostructure withrespect to the amount of a population, which is the sum of the amount ofthe immunoglobulin with a mannose-5 glycostructure, the immunoglobulinG(0) isoform, the immunoglobulin G(1) isoform, and the immunoglobulinG(2) isoform. With the method as reported herein the amount of theimmunoglobulin with a mannose-5 glycostructure is in one embodiment 10%or less of the population, in another embodiment 8% or less of thepopulation, and in a further embodiment 6% or less of the population.

The method as reported herein can be performed in certain embodiments ascontinuous cultivation, as fed-batch cultivation, or as combinationthereof, e.g. starting as fed-batch cultivation with subsequentcrossover to a continuous cultivation. Additionally, the method asreported herein can be performed in different ways. For example, in oneembodiment prior to the cultivating under conditions with a DGL valuebelow 1.0, i.e. for example under conditions wherein the availableamount of glucose is 80% or less of the amount of glucose that canmaximally be utilized by the cell in the culture per time unit, thecultivating is with an excess of glucose, i.e. a DGL value of 1.0. Inanother embodiment the cultivating is started with an amount of glucoseas contained in standard culture media, e.g. between 1 and 10 g/lculture medium, e.g. in order to obtain a predefined cell density, e.g.in one embodiment of 10⁵ cell/ml. In a further embodiment the startingof the cultivating is in the presence of an excess amount of glucose,i.e. a DGL of 1.0, and adding an amount of glucose per time unit, whichis 80% or less of the amount of glucose that can maximally be utilizedper time unit by the cells in the cultivation. In another embodiment thefeeding is started once the amount of glucose present in the culturemedium has dropped to or below a preset value in the cultivation. In thelast two cases the amount of glucose available in the culture is reducedby the metabolism of the cells in the cultivation.

In one embodiment the amount of glucose, which is available or added pertime unit and which is less than the amount of glucose that canmaximally be utilized, is kept at the same value, i.e. constant, in themethod as reported herein. For example, if an amount of 50% of theamount of glucose that can maximally be utilized per time unit isavailable, this amount is available in all time units of the method inwhich a restricted glucose feeding is performed. It has to be pointedout that this value is a relative value. Though, as the viable celldensity changes during the cultivation (i.e. it increases in thebeginning, reaches a maximum, and drops thereafter again) the absoluteamount of available glucose changes accordingly as it is a relativevalue depending on the absolute viable cell density. As the relativevalue is kept constant (i.e. at e.g. 80%) but the absolute referencevalue changes (i.e. e.g. increasing viable cell density) also therelative absolute value changes (i.e. 80% of an increasing value arealso increasing).

The term “per time unit” denotes a fixed time range, such as 1 minute, 1hour, 6 hours, 12 hours, or 24 hours. In one embodiment the time unit is12 hours or 24 hours. The term “amount of glucose available per timeunit” as used within this application denotes the sum of 1) the amountof glucose contained in the cultivation medium of a cultivation at thebeginning of a fixed time range and 2) the amount of glucose added, i.e.fed, during the time unit. Thus, an amount of glucose is added to thecell cultivation medium, e.g. to the cultivation vessel, which increasesthe amount of glucose in the cultivation medium at the beginning of thefixed time range to the predetermined amount. This amount of glucose canbe added, e.g., as solid, dissolved in water, dissolved in a buffer, ordissolved in a nutrient medium, whereby water and buffer shall notcontain glucose. The amount of glucose to be added corresponds to theamount of glucose to be available reduced by the amount of glucosepresent in the medium in the cultivation vessel. The process of addingthe amount of glucose can be performed either as single addition, asmultiple addition of small, equal fractions, or as continuous additionduring a time unit as described above.

The method as reported herein is suitable for any kind of cultivationand any cultivation scale. For example, in one embodiment the method isused for continuous or fed-batch processes; in another embodiment thecultivation volume is from 100 ml up to 50,000 l, in another embodimentfrom 100 l to 10,000 l. The method as reported herein is useful for theproduction of immunoglobulins with 10% or less, or 8% or less, or 6% orless of the immunoglobulin having a mannose-5 glycostructure. In oneembodiment the immunoglobulin is an immunoglobulin G or E. The method asreported herein comprises a eukaryotic cell, wherein the cell in turncomprises a nucleic acid encoding the heavy chain of an immunoglobulinor a fragment thereof and a nucleic acid encoding the light chain of animmunoglobulin or a fragment thereof. The eukaryotic cell is in oneembodiment selected from CHO cells, NS0 cells, BHK cells, hybridomacells, PER.C6® cells, Sp2/0 cells, HEK cells, and insect cells.

A person skilled in that art is familiar with medium compositions andcomponents as well as nutrient concentrations required by differentcells for optimal growth in addition to the amount of glucose and willchoose an appropriate medium for the cultivation of the cell (see e.g.Mather, J. P., et al. in Encyclopedia of Bioprocess Technology:Fermentation, Biocatalysis, and Bioseparation, Vol. 2 (1999) 777-785).

In one embodiment the amount of glucose that has to be available to thecells in a cultivation according to the method as reported herein iscalculated by multiplying the viable cell density, which can be achievednormally in the culture vessel at a certain point of time of thecultivation, with the volume of the culture vessel and the amount ofglucose that can maximally be utilized by the exponentially growingcells per time unit and by the intended DGL. In more detail, from thecourse of the glucose concentration in the cultivation and the course ofthe cell density in the cultivation prior to the actual point of timethe future course of the glucose concentration and the cell density arepredicted. With this prediction the amount of glucose that has to beadded to the cultivation to achieve the intended DGL is calculated withthe following formula:

(glucose  to  be  added [pg  glucose/ml/h]) = (current  cell  density  [cells/ml]) × (maximum  glucose  consumption  rate  of  the  cell  [pg  glucose/cell /h]) × (D G L  value) − amount  of  glucose  present  in  the  medium  in  the  cultivation  vessel.

In one embodiment the pH value of the cultivation is between pH 6.5 andpH 7.8. In another embodiment the pH value is between pH 6.9 and pH 7.3.In a further embodiment the pH value is between pH 7.0 and 7.2. It hasbeen found as outlined in Example 1 that in combination with arestricted glucose feeding with a pH value of 7.0 in the constantfeeding method the M5 content can efficiently be regulated to definedvalues, i.e. below 8%, compared to a pH value of 7.2. In thecultivations in the fed-batch method at pH values of 7.0 or 7.2,respectively, it was found that with the DGL control method the M5content could be regulated to be less than 5.5%. It has been found thatwith a reduction of the pH value of the cultivation an increase of theM5 amount due to the lowering of the DGL value can be traversed.

The cultivation is in one embodiment performed at a temperature between27° C. and 39° C., in another embodiment between 35° C. and 37.5° C.

With the method as reported herein any polypeptide containing aglycostructure can be produced, such as immunoglobulins, interferons,cytokines, growth factors, hormones, plasminogen activator,erythropoietin and the like.

The cultivating in the method as reported herein can be performed usingany stirred or shaken culture devices for mammalian cell cultivation,for example, a fermenter type tank cultivation device, an air lift typecultivation device, a culture flask type cultivation device, a spinnerflask type cultivation device, a microcarrier type cultivation device, afluidized bed type cultivation device, a hollow fiber type cultivationdevice, a roller bottle type cultivation device, or a packed bed typecultivation device.

The method as reported herein is performed in one embodiment for up to15 days. In another embodiment the cultivating is for 6 to 15 days. Inone embodiment the immunoglobulin is an anti-IL-6R antibody.

The method as reported herein is exemplified with an antibody to humaninterleukin-6 receptor as reported e.g. in EP 0 409 607, EP 0 628 639,U.S. Pat. No. 5,670,373, or 5,795,965 (herewith incorporated byreference in their entirety) as this antibody and the cell lineexpressing it were available at sufficient quantity in our laboratory atthe time of the invention. This is not intended to restrict the scope ofthe invention.

The following examples and figures are available to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

EXAMPLES

Materials and Methods

Cell Line:

An exemplary CHO cell line in which the amount of the mannose-5glycostructure of a recombinantly produced immunoglobulin can bemodified is a CHO cell line comprising a nucleic acid encoding ananti-IL-6 receptor antibody according to EP 0 409 607 and U.S. Pat. No.5,795,965. For the cultivation of the recombinant CHO cell any culturemedium can be used as long a glucose supplementation according to themethod of the invention can be performed. Exemplary culture media areIMDM, DMEM or Ham's F12 medium or combinations thereof, which have beenadapted to the method as reported herein in as much as the mass ratiosof the culture medium components to glucose are adopted. It is likewisepossible to exclude glucose from the cultivation medium and add it tothe cultivation separately.

Cultivation:

CHO cells expressing an anti-IL-6R antibody were cultivated in a 11 or21 fermentation vessel. The feeding medium contained 15 to 40 g/lglucose. Glucose could be fed with a separate concentrated solutioncontaining of e.g. 400 g/l glucose. The cultivation was performed at apH value of in the range from pH 7.0 to pH 7.2.

Determination of the Glycostructure:

For the analysis of IgG glycosylation pattern a method according toKondo et al. (Kondo, A., et al., Agric. Biol. Chem. 54 (1990) 2169-2170)was used. The IgG was purified from the centrifuged supernatant of thecultivation medium using a small scale protein A column. Theoligosaccharide of the purified IgG was released using N-glycosidase F(Roche Diagnostics GmbH, Mannheim, Germany) and labeled with 2-aminopyridine at the reducing terminus. The labeled oligosaccharide wasanalyzed by reverse-phase chromatography (HPLC). Each peak was assignedby both mass spectrometry and standards for the oligosaccharides.

Glucose Determination:

The glucose concentration was determined using an YSI 2700 SELECT™analyzer (YSI, Yellow Springs, Ohio, USA) with a method according to themanufacturer's manual.

Viable Cell Density Determination:

Viable Cell density was determined using an automatic image processingand analysis system (CEDEX®; Innovatis, Germany) and the trypan bluedye-exclusion method.

Example 1

Effects of the DGL Control and pH on Antibody Production and Mannose-5Glycostructure (M5) Content

A test was conducted using a CHO cell strain producing humanizedanti-human IL-6 receptor antibody (Tocilizumab, RoACTEMRA®)), which wasprepared in accordance with the method described in Referential Example2 of Japanese Unexamined Patent Publication No. 99902/1996 by use ofhuman elongation factor 1αpromotor as reported in Example 10 ofInternational Patent Application Publication No. WO 92/19759(corresponding to U.S. Pat. Nos. 5,795,965, 5,817,790, and 7,479,543).

In the constant absolute amount feeding method, effects of pH control onimmunoglobulin production were observed. Table 2 shows the effects of pHcontrol on antibody oligosaccharides production and M5 content inconstant feeding mode.

TABLE 2 Effects of pH control in constant absolute amount feeding mode.Relative antibody M5 Sample on pH set- concentration content No. [day]point DGL [%] [%] 1 7 7.0 0.80-0.45 90.1 3.6 2 7 7.0 0.49-0.21 100 5.4 37 7.2 0.73-0.35 135.1 11.7 4 7 7.2 0.69-0.30 120 10.8 5 7 7.2 0.35-0.29127 25.2 6 7 7.2 0.64-0.25 122.5 8.7

At pH 7.0 the amount of the mannose-5 glycostructure (M5) was regulatedto less than 5.5%. The DGL value declined from 0.80 to 0.21 due to thechange of cell density. On the other hand, at pH 7.2, the M5 amountfluctuated between 8.7% and 25.2% and was higher than that at pH 7.0.The DGL value at pH 7.2 varied from 0.73 to 0.25. Moreover, in thiscase, immunoglobulin production at pH 7.2 was more than 120% (relativevalue compared to pH 7.0). Higher immunoglobulin production in theconstant absolute amount feeding method induces a higher M5 content ofmore than 8%. Therefore, with a pH 7.0 control in the constant absoluteamount feeding method the M5 content could efficiently be regulated tolower values, i.e. below 8%, compared to pH 7.2 control method.

The DGL control method (=constant relative amount feeding method) wasalso used for the immunoglobulin production by fed-batch mode at variouspH values, and the M5 content was analyzed. Table 3 shows the effects ofDGL control after the start of feeding at day 2-3 and pH onimmunoglobulin production and M5 content.

TABLE 3 Effects of DGL and pH control in fed-batch mode. Relative Sampleantibody M5 on pH set- concentration content No. [day] point DGL [%] [%]1 7 7.0 0.8 102.7 2.9 2 7 7.0 0.6 96.2 2.7 3 7 7.0 0.4 100.0 3.3 4 7 7.00.3 91.1 3.9 5 7 7.0 0.2 83.0 4.0 6 7 7.2 0.6 100.9 4.4 7 7 7.2 0.4 90.15.3

At pH 7.0 the DGL control method was applied in the range of a DGL from0.2 to 0.8. As a result, the M5 content was regulated to be equal orless than 4.0%. On the other hand, at pH 7.2, the DGL value was operatedin the range from 0.4 to 0.6. Here the M5 content could be controlled tobe less than 5.5%.

Example 2

Cultivating with Different DGL Values

The cultivating of a CHO cell comprising a nucleic acid encoding ananti-IL-6R antibody was performed with different DGL values. The resultsare summarized in the following Table 4.

TABLE 4 Effects of DGL control value on immunoglobulin production and M5content. Relative M5 G(0) G(1) Sample antibody con- con- con- G(2) onconcentration tent tent tent content No. [day] DGL [%] [%] [%] [%] [%] 17 0.6-0.5 107.3 3.5 38.4 46.7 11.4 2 7 0.4 111.0 3.5 38.8 46.9 10.8 3 70.2 111.5 4.5 40.1 45.2 10.1 4 8 const. 100.0 5.9 43.8 42.0 8.3 feeding

Compared to a constant feeding shows the controlled DGL strategy with aDGL value of 0.4 to 0.6 a reduced mannose-5 content.

Example 3

Cultivating with Different Feeding Strategies

The cultivating of a CHO cell comprising a nucleic acid encoding ananti-IL-6R antibody was performed with one DGL value but with differentfeeding strategies. The results are summarized in the following Table 5.

TABLE 5 Effects of feed strategy on viability and viable cell density.viable cell Sample viability density [×10⁶ No. after [h] DGL feedingadjustment [%] cells/ml] 1 112 0.4 single daily 71 5.1 2 115 0.4 dualdaily 75 5.8 3 115 0.4 single profile 73 4.9 4 115 0.4 dual profile 705.1

In the single feed experiments a single feed was used containing allnutrients and glucose. In the dual feed experiments two feeds were used:the first feed contains all nutrients and glucose at a low concentrationof 15 g/l and the second feed contains a high concentration of glucose.These different feed experiments were performed in one set with a dailyadjustment of the feeding rate and in another set following apredetermined profile based on the viable cell density developmentrecorder in earlier cultivations. As can be seen from Table 5 viabilityand viable cell density are comparable independently of the employedfeeding strategy.

Example 4

Degree of Glucose Limitation (DGL) Control for Immunoglobulin Productionby the Fed-Batch Mode

CHO cells (8.0−12×10⁵ cells/ml) were inoculated in serum free culturemedia as described above. The cells were grown at 37° C., 98% relativehumidity, and 10% CO₂ atmosphere. In the fed-batch cultivation thefeeding medium containing glucose was started to be fed to the mainfermenter on the 2^(nd) or 3^(rd) day from the beginning of thecultivation. The feeding strategy followed the method to control thedegree of glucose limitation (DGL) according to U.S. Patent ApplicationPublication No. US 2006/0127975 A1. The DGL can be defined as the ratioof the observed specific glucose consumption rate to the maximum knownspecific glucose consumption rate when glucose is freely available forthese cells (DGL=Q(glc)/Q(glc)_(max), where Q(glc)=currently observedspecific glucose consumption rate; Q(glc)_(max)=maximum known specificglucose consumption rate for these cells).

FIG. 1 shows the viable cell density and cell viability profiles of thecultivation. The DGL was controlled to be at a value of 0.4-0.5 invarious cell densities as shown in FIG. 2. The feeding rates werechanged once or twice a day depending on the cell density at that time.FIG. 3 shows the feeding profiles based on DGL by the fed-batch mode.The feeding rate was changed between 0.8 and 1.6 ml/h depending on thecell density. With this feeding strategy applied, an immunoglobulinproduction profile was obtained as shown in FIG. 4. Using theinoculation size of 10×10⁵ cells/ml and 12×10⁵ cells/ml, theimmunoglobulin production was almost the same and more than 120% of theimmunoglobulin production in constant feeding method at day seven asshown in Table 6 (feeding rate of 0.02 g glucose/h). In spite of the 20%difference in the initial cell densities, it was possible with the DGLcontrol method to obtain approximately equivalent immunoglobulin titer.Moreover, when the inoculation size was set at 8.0×10⁵ cells/ml, despitethe 20 hour delay of the feeding start point, the immunoglobulinobtained was more than 110% (relative value) at day seven. In theseresults, the DGL control method could achieve a stable immunoglobulinproduction at various inoculation sizes.

Example 5

The Effects of the DGL Control on the Mannose-5 Glycostructure andGalactosylation of Oligosaccharides

Of the immunoglobulin produced by fed-batch cultivation using the DGLcontrol the glycosylation pattern was analyzed. Table 6 shows the resultof the oligosaccharide analysis for the immunoglobulin obtained from theDGL controlled fed-batch cultivation in comparison with the constantfeeding method (feeding rate: 0.02 g of glucose/h). At the inoculationsize of 8.0×10⁵ cells/ml, the content of mannose-5 glycostructure (M5)was 2.8%. At the inoculation size of 10×10⁵ cells/ml and 12×10⁵cells/ml, the M5 content was 4.1% and 3.8%, respectively. At allcultivation conditions, the DGL control method was able to regulate theM5 content to less than 5.0%.

Meanwhile, in each condition, immunoglobulin G(0) isoform andimmunoglobulin G(2) isoform were controlled at the range from 40% to 46%and from 9.0% to 11%, respectively.

TABLE 6 Effects of DGL control value on immunoglobulin production andglycosylation pattern. relative Sample inoculation antibody M5 G(0) G(1)G(2) on cell density concentraction conten content content content No[day] DGL [×10⁵ cells/ml] [%] [%] [%] [%] [%] 1 7 constant 10 100.0 3.545.7 41.5 9.2 feeding 2 7 0.4 8 112.5 2.8 41.7 44.7 10.8 3 7 0.4 10122.6 4.1 42.9 43.1 9.8 4 7 0.4 12 127.1 3.8 45.5 41.5 9.1

What is claimed:
 1. A method for the production of an immunoglobulincomprising a) cultivating a Chinese Hamster Ovary (CHO) cell comprisinga nucleic acid encoding the immunoglobulin in a cultivation medium withrestricted glucose feeding wherein the amount of glucose available inthe cultivation medium per time unit is kept constant in all time unitsin which restricted glucose feeding is performed and the degree ofglucose limitation (DGL) is limited to a single constant value in arange between 0.5 and 0.1, wherein feeding is started once the amount ofglucose present in the cultivation medium has dropped to or below apreset value in the cultivation and wherein said cultivation is afed-batch cultivating, and b) recovering the immunoglobulin.
 2. Themethod according to claim 1, wherein the restricted glucose feeding isstarted at day 2 or day 3 of the cultivating.
 3. The method according toclaim 1, wherein the cultivating is at a pH value from pH 6.5 to 7.5. 4.The method according to claim 3, wherein the cultivating is at a pHvalue from pH 6.9 to 7.3.
 5. The method according to claim 4, whereinthe cultivating is at a pH value from pH 6.95 to pH 7.05 or at a pHvalue from pH 7.15 to pH 7.25.
 6. The method according to claim 1,wherein the immunoglobulin is an immunoglobulin of class G or class E.7. The method according to claim 1, wherein cultivating the CHO cell isperformed for six to twenty days.
 8. The method according to claim 1,wherein the immunoglobulin is an anti-IL-6R antibody.
 9. The methodaccording to claim 1, wherein the DGL is limited to a constant value ina range between 0.45 and 0.2.
 10. The method according to claim 1,wherein the DGL is limited to 0.4, or 0.2.
 11. The method according toclaim 8, wherein the anti-IL-6R antibody comprises tocilizumab.
 12. Themethod according to claim 8, wherein the DGL is limited to a constantvalue in a range between 0.45 and 0.2.
 13. The method according to claim8, wherein the DGL is limited to 0.4, or 0.2.
 14. The method accordingto claim 11, wherein the DGL is limited to a constant value in a rangebetween 0.45 and 0.2.
 15. The method according to claim 11, wherein theDGL is limited to 0.4, or 0.2.
 16. The method according to claim 1,wherein the fraction of the immunoglobulin with a mannose-5glycostructure is 10% or less of the sum comprising the amount of thepolypeptide with a mannose-5 glycostructure, the amount of thepolypeptide G(0) isoform, the amount of the polypeptide G(1) isoform,and the amount of the polypeptide G(2) isoform after 7 days ofcultivation.
 17. The method according to claim 1, wherein the fractionof the immunoglobulin with a mannose-5 glycostructure is 6% or less ofthe sum comprising the amount of the polypeptide with a mannose-5glycostructure, the amount of the polypeptide G(0) isoform, the amountof the polypeptide G(1) isoform, and the amount of the polypeptide G(2)isoform after 7 days of cultivation.
 18. A method for the production ofan immunoglobulin comprising a) cultivating a Chinese Hamster Ovary(CHO) cell comprising a nucleic acid encoding the immunoglobulin in acultivation medium with restricted glucose feeding wherein the amount ofglucose available in the cultivation medium per time unit is keptconstant in all time units in which restricted glucose feeding isperformed and the degree of glucose limitation (DGL) is limited to asingle constant value in a range between 0.5 and 0.1, and wherein therestricted glucose feeding is started at day 2 or day 3 of thecultivating and wherein said cultivating is a fed-batch cultivating, andb) recovering the immunoglobulin.
 19. The method according to claim 1,wherein the time unit is about 1 minute.
 20. The method according toclaim 1, wherein the time unit is about 1 hour.
 21. The method accordingto claim 1, wherein the time unit is about 6 hours.
 22. The methodaccording to claim 1, wherein the time unit is about 12 hours.
 23. Themethod according to claim 1, wherein the time unit is about 24 hours.24. The method according to claim 1, wherein density of CHO cells at thebeginning of the cultivating is about 10⁵ cells per milliliter.
 25. Themethod according to claim 1, wherein density of CHO cells at thebeginning of the cultivating is about 8×10⁵ cells per milliliter. 26.The method according to claim 1, wherein density of CHO cells at thebeginning of the cultivating is about 10×10⁵ cells per milliliter. 27.The method according to claim 1, wherein density of CHO cells at thebeginning of the cultivating is about 12×10⁵ cells per milliliter. 28.The method according to claim 1, wherein density of CHO cells is fromabout 10⁵ cells per milliliter to about 60×10⁵ cells per milliliterduring the cultivation.
 29. The method according to claim 18, whereinthe time unit is about 1 minute.
 30. The method according to claim 18,wherein the time unit is about 1 hour.
 31. The method according to claim18, wherein the time unit is about 6 hours.
 32. The method according toclaim 18, wherein the time unit is about 12 hours.
 33. The methodaccording to claim 18, wherein the time unit is about 24 hours.
 34. Themethod according to claim 18, wherein density of CHO cells at thebeginning of the cultivating is about 10⁵ cells per milliliter.
 35. Themethod according to claim 18, wherein density of CHO cells at thebeginning of the cultivating is about 8×10⁵ cells per milliliter. 36.The method according to claim 18, wherein density of CHO cells at thebeginning of the cultivating is about 10×10⁵ cells per milliliter. 37.The method according to claim 18, wherein density of CHO cells at thebeginning of the cultivating is about 12×10⁵ cells per milliliter. 38.The method according to claim 18, wherein density of CHO cells is fromabout 10⁵ cells per milliliter to about 60×10⁵ cells per milliliterduring the cultivation.
 39. The method according to claim 1, whereincultivating the CHO cell is performed for six to fifteen days.
 40. Themethod according to claim 1, wherein cultivating the CHO cell isperformed for six to eight days.
 41. The method according to claim 1,wherein the fraction of the immunoglobulin with a mannose-5glycostructure is 8% or less of the sum comprising the amount of thepolypeptide with a mannose-5 glycostructure, the amount of thepolypeptide G(0) isoform, the amount of the polypeptide G(1) isoform,and the amount of the polypeptide G(2) isoform after 7 days ofcultivation.